A USDA-ARS Project to Evaluate Resistance to Varroa jacobsoni by Honey Bees of Far-Eastern Russia

Apis mellifera is not native to the Primorsky Territory on Russia's Pacific coast, but was first moved there in the last century. At that time, pioneers from western Russia took advantage of the completion of the Trans-Siberian Railway and moved bees from European western Russia to the Primorsky Territory in Asian far-eastern Russia. This far-eastern area of Russia is within the natural range of Apis cerana, the original host of Varroa jacobsoni. Thus A. mellifera was brought into the likely range of V. jacobsoni even before the parasite was scientifically described in 1904. This probable long association of V. jacobsoni and A. mellifera in the region has engendered one of the best opportunities in the world for A. mellifera to develop genetic resistance to V. jacobsoni.

The USDA-ARS Honey Bee Breeding, Genetics & Physiology lab, Baton Rouge, LA, explored whether such resistance might be found in Primorye populations of honey bees in the autumn of 1994. Data were collected showing that colonies in the area did have Varroa infestation, but that the levels were lower compared to U.S. colonies.

The experimental yard in Khorol, Primorsky, approximately 200 km NW of Valdivostock

In June 1995, a test apiary was established in Primorsky. Queens from a variety of sources in the territory were introduced into 50 colonies. Mite levels were equalized among colonies, and thereafter no treatments for mite control were undertaken. Between August 1995 and September 1996, monthly infestation rate data for worker and drone brood were collected. For comparison, we ran a test in Baton Rouge, collecting similar data.

Russian cooperators, among them Dr. Victor Kusnetsov (right) and Anatoly Reshetnikov (second from left), the beekeeper who managed the experimental colonies used in the project.

Worker brood infestation remained quite low in the Primorsky bees. Even 15 months after the last treatment, the average infestation was only 7%. In the U.S. , 12 months after treatment, the average infestation was 33% and many of the colonies were collapsing with "parasitic mite syndrome". In the summer of 1996, the average infestation in the U.S. colonies rose substantially, but did not rise in the Russian colonies.

A similar difference occurred on drone brood infestations. In colonies in both areas Varroa infestations were higher in drones.In Russian colonies, the highest average infestation of 39% occurred in June 1996 and average infestations declined after thereafter.In U.S. colonies, infestation rates began at 37% and continued to rise to an average of 76% in August 1996.At this time the colonies were treated with Apistan in order to keep them from dying.

An Importation of Potentially Varroa-Resistant Honey Bees from Far-Eastern Russia

In an earlier report (ABJ 135:11, 746-748) we described the initiation of a project to evaluate potential resistance to Varroa jacobsoni by honey bees from the Primorsky Territory on Russia's Pacific coast.

Because the two sets of data were not collected in the same place under the same conditions, a direct comparison of the data from Russia and the data from the United States cannot be used to conclude that the Russian bees showed or did not show resistance to Varroa. The only sure way to determine if the encouraging results we saw in the Primorsky were due to genetic resistance was to test the bees in the U.S. with U.S. mites.

In late June of 1997, a collection of 100 Primorsky honey bee queens was made and brought to the U.S. for further research. These queens were obtained from 16 separate beekeepers from a variety of places in the Primorsky. Some queens were grafted by beekeepers in preparation for providing queens. The collection of open-mated queens represented a total of 57 queen mothers. From the previous experiment, 2 queens which produced colonies having the lowest rates of infestation in the trial were each used to produce 10 daughter queens.

Below: Boats from the Louisiana Department of Wildlife and Fisheries helped transport the colonies to Quarantine station on Grand Terre Island.

Above: Dr. Victor Kuznetsov (fourth from left), and Dr. Bob Danka (second from right) with some of the cooperating beekeepers in Primorsky.

The queens were brought to the USDA, ARS, Honey Bee Quarantine Station at Grand Terre Island, Louisiana, on July 1, 1997 and installed into colonies prepared for them. The introduction was monitored by APHIS and the Louisiana Department of Agriculture and Forestry. As the colonies developed they were monitored for the presence of disease or parasites. Some colonies in the apiary, headed by queens of four commercial United States stocks, remained in the apiary as an additional check for the presence of slowly developing diseases.

Below: The Russian Queens are inspected one more time before introduction.

Evaluations of the Varroa-resistance of Honey Bees Imported from Far-Eastern Russia

Results from a natural history study of Varroa jacobsoni infestations in Apis mellifera in the Primorsky territory of Russia led to the importation of 100 queen honey bees into the United States in 1997. Early in February 1998, the queens and their colonies were moved to secure apiaries near the USDA, ARS, Honey Bee Breeding, Genetics and Physiology Laboratory in Baton Rouge, Louisiana, and lab personnel to began research on the potential resistance to V. jacobsoni.

The Russian colonies were treated to reduce mite populations below detectable levels. At the same time Varroawere being reared in domestic colonies to provide a source of inoculum mites for the test.

A bulk package of approximately 70 lbs of bees was made up from the domestic source colonies, then divided into smaller inoculation packages containing an average 1,797 worker bees with 1 mite per 6.9 bees.

Inoculation packages were introduced into the Russian and the control colonies.

Sticky board traps were placed on the bottom boards of the colonies to catch any falling mites.

Ten days after inoculation, the boards and cages were removed and the bees, remaining mites in the cages and mites on the sticky boards were counted. These counts were used to estimate the total number of mites that successfully entered each colony from the inoculation cage. After 9 weeks, the total number of mites in each colony was estimated from examination of worker brood, drone brood and adult workers. The sum of the estimates of mites in the worker brood, drone brood and adult bees provided an estimate of the number of adult mites in the colony.

The population growth of mites in each colony was calculated, and this growth was termed the "Mite Index". This is an expression of the number of times the mite population has increased onefold. The expected mite index was determined by using a mathematical model (Fuchs, Apidologie 1990).

The distribution of colonies according to their mite index. "11.4" is the mite index calculated from a mathematical model for colonies not having resistance to Varroa jacobsoni.

The distribution according to mite index of colonies selected to continue a breeding program and colonies not selected for the breeding program.

Based on the results of this test, forty of the queens that were imported from Russia were chosen as "breeders" to found a stock of resistant honey bees and to produce offspring for further tests.

While the test of the colonies produced by the imported queens was being conducted, general observations were made on a weekly basis concerning the honey production, general vigor and disease status. Although the mite index for each colony was an important factor determining whether or not a colony would be used in stock formation, these other factors also contributed.

The data suggested at least one mechanism of resistance was expressed. Non-resistant colonies had 65-75% brood infestation, while the chosen breeders had an average of 48.1%. Hence, lower rates of brood infestation will lead to overall lower rates of population increase for mites.

Future research with the Russian stock will focus on large multi-state field trials. The field trials are intended to supply information concerning comparative V. jacobsoni resistance and comparative honey production of the Russian stock in commercial beekeeping. Favorable results from these trials will lead to a general release of the stock to the beekeeping industry.

Resistance to the Parasitic Mite Varroa destructor in Honey Bees from Far-Eastern Russia

Varroa destructor is a parasitic mite of the Asian honey bee Apis cerana. Owing to host range expansion, it now plagues Apis mellifera, the world's principal crop pollinator and honey producer. Evidence from A. mellifera in far-eastern Russia, Primorsky (P) originating from honey bees imported in the mid 1800's, suggested that many colonies were resistant to V. destructor. A controlled field study of the development of populations of V. destructor shows that (P) colonies have a strong, genetically based resistance to the parasite. As control colonies (D) were dying with infestations of ~ 10,000 mites, (P) colonies were surviving with infestations ~ 4,000 mites. Several characteristics of the (P) bees contributed to suppressing the number of mites parasitizing their colonies.

Overall, Primorsky (P) honey bees appear to have several mechanisms which act in concert to provide them with substantial resistance to V. destructor. It is unlikely that we have yet identified all of the factors that may contribute to this resistance. Indeed, a substantial number of hypothesis remain wholly or partially untested. However, the the diversity of traits identified in this study that may contribute to the resistance suggests that a constellation of traits and genes underlie the overall resistance and provide opportunities for further development of the resistance through selective breeding.

Field trials of Russian honey bees (ARS Primorsky stock) propagated as queen lines from queens imported from the far-eastern province of Primorsky were conducted in 1999 and 2000 in Iowa, Louisiana, and Mississippi. Varroa destructor populations in Primorsky colonies grew more slowly and hence, had fewer numbers than they did in domestic colonies. Colonies of six Primorsky queen-lines evaluated in 1999 averaged about half the number of mites found in domestic control colonies. In 2000, colonies of 10 Primorsky queen lines in Louisiana supported an average V destructor population growth of 2.5 fold increase across 91 days, far less than the 17.3 fold increase predicted from growth models derived for domestic colonies. Most colonies of the same 10 Primorsky queen-lines in Iowa and Mississippi had no (150 colonies) to very few (48 colonies) detectable V destructor three months after being inoculated with about 100 mites. Hence, in all trials, ARS Primorsky honey bees showed strong resistance to V. destructor. Variance within and between queen lines indicates good potential to further increase this resistance through selective breeding.

Fig.1 - The average number of adult V. destructor mites in Primorsky (Blue) and domestic (White) honey bee colonies at 4 examinations separated by 34 day intervals in three states in 1999. i, an inoculation of about 100 mites was given to colonies in Louisiana and Mississippi at the time indicated.

Fig. 2 - The average mite population growth (MPG) expressed as fold increase in V. destructor mite populations in Primorsky (Blue) and domestic (White) honey bee colonies at 4 examinations separated by 34 day intervals in three states in 1999. Period 1 (not shown) provided baseline data.

Field trials of Russian honey bees (ARS Primorsky stock) propagated as queen lines from queens imported from the far-eastern province of Primorsky were conducted in 1999 and 2000 in Iowa, Louisiana, and Mississippi. While honey production varied between apiaries and states, the honey production of the majority of Primorsky queen lines met or exceeded commercial standards. For example, the best production came from Mississippi in 2000. There, the overall average production was 125 pounds, not including fall production. Selected breeder queens from Mississippi in 2000 averaged 185 pounds and ranged from 149 to 238 pounds. Overall, given favorable nectar flows and beekeeping, ARS Primorsky stock, selected for retention in the breeding program and released to the beekeeping industry, will not sacrifice honey production.

Fig. 2 - Relative honey yield of individual ARS Primorsky honey bees selected as breeder queens in 1999 to produce daughters for stock propagation or release and the average honey yield of domestic control colonies. The Z-score or relative rank in comparison to group average for Primorsky and domestic colonies in apiaries was used rather than absolute honey production. This permits the comparison of colonies or groups of colonies in different states and apiaries. The dark circles indicate queens selected for breeding based on both honey production and resistance to V. destructor. The rectangle indicates the average for domestic control colonies. Queen lines with no indicated breeder queen have been culled from the program.

Fig. 4 - Relative honey yield of individual ARS Primorsky honey bees selected as breeder queens in 2000 to produce daughters for stock propagation or release and the average honey yield of domestic control colonies. The Z-score or relative rank in comparison to group average for Primorsky and domestic colonies in apiaries was used rather than absolute honey production. This permits the comparison of colonies or groups of colonies in different states and apiaries. The dark circles indicate queens selected for breeding based on both honey production and resistance to V. destructor. The rectangle indicates the overall average for all colonies. Queen lines with no indicated breeder queen have been culled from the program.

ARS Primorsky honey bees were evaluated for their resistance to Acarapis woodi by monitoring natural infestations in colonies located in Iowa, Louisiana and Mississippi. In 1999, Primorsky colonies had lower levels of A. woodi infestation than the domestic colonies. Low tracheal mite infestations were also observed in 2000. The 10 Primorsky queen-lines tested were all resistant to tracheal mites. However, resistance was more pronounced in Louisiana and Mississippi wherein 0-3% infestations were recorded. It is possible that environmental factors influenced the degree of infestation between the states and within Primorsky lines, especially in Iowa. With careful selection, it is possible to further enhance resistance of Primorsky honey bees to tracheal mite parasitism.

It is important to understand that the release program is held captive by the laws of honey bee genetics.These laws dictate that the release program will require two years to make full Russian colonies widely available.In the first year of the release (2000), the Russian production queens that were being sold had been mated to domestic drones that were produced by the various queen breeders.Colonies produced by these Russian queens are hybrid colonies.We cannot predict the Varroa resistance or honey production of all the different hybrids that will be produced in the country.The second year (2001), queen breeders will be able to produce Russian drones to mate with their Russian queens, so beekeepers then will be able to buy Russian queens that will produce "pure" Russian colonies.

This paper (The Release of ARS Russian Honey Bees), should help clarify this and other questions about what the Russian bees are and what they are not.

Based on the findings of the resistance test, 40 Russian queens were selected to produce daughter queens for two extensive field trials in which the resistance of the Russian honey bees to V. jacobsoni was directly compared to that of domestic honey bees commonly used commercially in the United States.One trial began in mid-summer of 1998 and ended in early December 1999.The other trial was conducted in the spring and summer of 1999 in commercial apiaries in Iowa, Louisiana, and Mississippi, in cooperation with Mr. Manley Bigalk, Mr. Steven Bernard and Mr. Hubert Tubbs, respectively. All of these gentlemen are commercial beekeepers who are primarily honey producers.InAugust of 1999, when most of that year's data were collected, ARS held a teleconference with these beekeepers.These beekeepers had seen the bees in their own apiaries and could evaluate the quality of the stock for general beekeeping characteristics as well as help to evaluate the commercial implications of the data.The beekeepers recommended that a procedure to release the Russian honey bees begin.

The release program is not a one time event.Rather, it is a cycle of releases resulting from an underlying stock maintenance and selection program (see the figure at left).The queens that produced the colonies having the most resistance to V. jacobsoni, and were otherwise acceptable for commercial beekeeping, were divided into three groups or "blocks".For the purposes of maintaining the stock, daughters of the queens of each of the three blocks are mated with drones produced by queens from the other two blocks. For purposes of selection in 1999, the queens in block "A" were used to produce daughters for the large field trial with cooperating beekeepers.

Each of 6 queen lines was represented by daughter queens in each state and each apiary.As a result of the field trials, one queen line was dropped from the program, two queen lines were maintained in the program but not used to produce breeder queens for distribution, and the best queen in three of the lines was selected to produce breeder or queen line queens for distribution to the industry.

An isolated island mating station was used in order to have natural matings of the desired combinations of queens and drones.We chose natural matings in order to maintain the high genetic diversity afforded by queens mating with about 20 drones if allowed to naturally mate.

RIGHT: The conservation camp at the mouth of Bird Island bayou can be seen on Marsh Island.

Of the five queen lines retained by the program, the best daughter was used to propagate daughters for stock maintenance.In 2000, queen lines from the 1997 importation which had been assigned to the other two blocks were evaluated in similar selection procedures to identify the program breeder queens.Thus, the program has provisions for selection between several queen lines each year.Also, the program will select the best queens of the selected lines.These queens will be used to both propagate the queen lines and propagate breeder queens for the industry.

The Honey Bee Breeding, Genetics & Physiology Laboratory entered into another cooperative agreement with the Russian Academy of Sciences.Under this agreement, initial screening of queens in Russia will be done each year for three more bee seasons.Each year, the best of the queens studied in Russia will be brought to the United States for further study and possible incorporation into one of the three queen line blocks, This program is underway.In June of 1999 and July2000, an additional 120 queens were collected and brought to the United States.As before, these queens went to the island quarantine facility.The 1999 queens were screened for resistance to V. jacobsoni during the 2000 bee season, and the 2000 queens will be screened during the 2001 bee season.Also, laboratory staff will be evaluating other queens from Russia in addition to those that are screened for the program by Russian scientists.By the end of this portion of the program, the colonies produced by between 500 and 700 queens will have been studied and evaluated as potential sources of new queen lines for the breeding and selection program in the next 5 to 8 years.

Because of the continuing flow of new queens from Russia into the program and our selection within and between the existing queen lines, we will be able to both apply strong selection for resistance to V. jacobsoni and still be able to maintain good genetic diversity within the overall stock.The main criterion for selection will be resistance to V. jacobsoni.However, resistance to Acarapis woodi (tracheal mites), honey production, and the presence of chalkbrood in colonies will also be considered when we make selections of the parents of future generations.Because of these aspects of the program, the ARS Russian honey bee stock will be different from year to year.We expect that the most noticeable difference will be a slow, steady increase in the expression of desirable traits.

In the first year of the release program (2000), those who obtained production queens were getting Russian queens that produced hybrid colonies.Because of the genetic nature of honey bees, the Russian queens of the first year produced hybrid worker bees, but also produced Russian drones.These drones will be available for matings the second year (2001).During the first year, it was recommended that beekeepers (both queen breeders and those that bought production queens) produce or obtain enough hybrid Russian colonies to produce the drones needed for the second year to produce "pure" Russian matings.Queen breeders will need the Russian drones to produce Russian queens that will foster "pure" Russian colonies.General beekeepers who had hybrid colonies the first year will have a supply of Russian drones that will mate with any Russian supersedure or other queens that may be produced in their apiaries.They will thereby assure that their apiaries with ARS Russian stock will remain Russian.

It was not possible for the laboratory to test all of the various hybrids that were produced in the spring of 2000.We expect that at least some of these hybrids would be quite desirable.In such instances, queen breeders might consider organizing at least some of their program to continue to offer queens that will produce hybrid colonies.However, our best prediction is that there will probably be large variations between different types of hybrids, but generally, that they will be within the usual range for commercial honey bees in the United States, which also are highly varied.

Each year, the program will release queens that are sufficiently unrelated to the prior year's queens that inbreeding will be avoided.Of course, people are welcome to organize their own breeding and selection programs that include Russian honey bees.Indeed, we encourage breeders to include some ARS Russian honey bee parentage into their own programs in order to enhance resistance to V. jacobsoni in their own stocks of honey bees.However, attempting to produce ARS Russian honey bee stock by making crosses other than those recommended by the program may result in inbreeding problems.Also, such stock will not have the advantages to be derived from the ongoing selection program.

The current releases of Russian honey bee breeder queens are resistant to V. jacobsoni.This resistance is strong enough to have economic value.Beekeepers should be able to use half as many treatments for the control of V. jacobsoni as they are currently using.The ARS Russian honey bees are not immune to V. jacobsoni.Given enough time, many of them will succumb to the mites.However, ARS Russian honey bees are a good centerpiece for integrated pest management approaches to the control of V. jacobsoni that rely much less on miticides.As the selection program proceeds, the level of resistance to V. jacobsoni in the ARS Russian honey bees predictably will be further improved, and the need for miticides will be further reduced.

The ARS Russian honey bee stock is not "finished".Indeed, selection will be continuing for at least several years.Because of this, the ARS Russian honey bee stock will be continually changed and improved for the life of the selection program.This selection program is designed to produce Russian honey bees of the future that will contribute to the gene pool of the honey bees in the United States in two ways.First, it is and will be developed as a stock in its own right.Second, it will be bred in ways that it will be useful as a source of genetic material to enhance existing stocks of honey bees, especially in regard to resistance to both V. jacobsoni and Acarapis woodi.Hence, ARS Russian honey bees, and the breeding program to further improve them, are a resource for all of American beekeeping.

Hygienic behavior contributes to the overall disease resistance of honey bee colonies. It is the detection and removal of diseased or mite-infested brood from a colony. This study evaluated the hygienic behavior of domestic and ARS Primorsky honey bees. The Primorsky honey bees are found to be more hygienic than the domestic colonies. This observation suggests that the overall disease and pest resistance of Primorsky bees is quite good. The use of Primorsky bees will enhance the profitability of commercial beekeeping by reducing disease control costs.

The removal rate of colonies was determined by using liquid nitrogen, and freezing a 3-inch diameter circular section of capped worker brood enclosing approximately 300 cells. Using a digital camera, test sections were photographed before liquid nitrogen was poured. Test sections were also mapped on plastic sheets to facilitate identifying them. Brood frames with the frozen sections were returned and placed in the center of the brood nest of their respective colonies for 48 hours. After removal from the colony the test sections were photographed again. The before and after freezing photographs were compared and the capped and uncapped cells counted. The number and percentage of cells that were subject to complete hygienic behavior were then calculated.

Liquid Nitrogen is being poured into cylinders (cans with both ends cut out) placed over the selected capped worker brood

A close up shows the cylinder while the liquid nitrogen evaporates.

Results from two assays showed that Primorsky honey bees consistently removed more dead brood than the domestic colonies. For both assays, 41% of the Primorsky honey bee colonies tested were considered hygienic (> 95% dead bee removal). Only 21% of the domestic colonies showed the hygienic trait. No correlation between removal rate and adult bee population was observed.

Tracheal mites, Acarapis woodi, are parasites that pose a significant health problem for honey bees in the United States and in many other countries. Fortunately, some stocks of bees have an inherent genetic resistance to being infested by mites. Until the experiments described here, it was not known what specific characteristics enabled bees to resist infestation by the mites.

Tracheal mites in a honey bee trachea (photo at 30X)

We tested the possibility that resistant bees may be better able to groom mites from their bodies. Bees of two stocks, one resistant and one susceptible to mites, were compared when we impaired their ability to groom themselves. Responses of both stocks were tested by removing the middle legs of uninfested, young adult bees, exposing the bees to mites in infested colonies, then retrieving the test bees and measuring parasitism.

Worker bee grooming its thorax with the right middle leg

In both stocks, bees with middle legs removed had greatly increased mite abundances. Most importantly, resistant bees with legs intact were much more efficient at removing mites than susceptible bees were. This is evidence of the importance of grooming as a mechanism of resistance. Mite infestation increased as more (0 vs. 1 vs. 2) middle legs were removed. In bees with only one leg removed, mite infestations were greater on the treated side. Restraining rather than removing middle legs also resulted in increased infestation.

The results provide the first evidence of how some bees are able to avoid mite infestation. The findings are potentially useful to scientists and bee breeders trying to identify other resistant stocks and in selecting for the economically important trait of resistance.

Genetic resistance to tracheal mites can be used by bee breeders to improve stocks

Reference to full article:

Evidence of autogrooming as a mechanism of honey bee resistance to tracheal mite infestation. Journal of Apicultural Research 37: 39-46 (1998) by R. G. Danka and J. D. Villa.

Well Groomed Bees Resist Tracheal Mites (1998)

A Survey of Tracheal Mite Resistance Levels in U.S. Commercial Queen Breeder Colonies

Research has shown that honey bee strains having genetic resistance to tracheal mites (Acarapis woodi) can be used to help to solve the problems resulting from parasitism by this mite. Colonies of resistant bees tend to withstand challenge from tracheal mites and remain productive without treatment, while susceptible colonies tend to become infested at damaging levels.

Resistance to tracheal mites is of interest to bee breeders who are trying to improve stock quality. Identifying the level of resistance in breeding colonies should be a critical part of such breeding efforts. However, little is currently known about the resistance in the commercial breeding population used to supply queens for the U.S. beekeeping industry. Our objectives were to measure the range of resistance in a sample of U.S. commercial breeder colonies, and enable participating queen breeders to improve the quality of their stock by providing selection guidance.

Eight commercial queen producers from five states submitted brood from 6 to 19 breeder colonies each so that emerging bees could be evaluated for relative resistance to tracheal mites. Young, uninfested bees from each colony of an individual queen producer, and also from colonies of two reference stocks (one known to be resistant to tracheal mites and one known to be susceptible), were marked and then simultaneously exposed to mites in infested colonies. They were retrieved after 4-6 days and dissected to determine resulting mite infestations. Results for the breeder colonies were adjusted to the average results of the resistant and susceptible reference colonies with which they were tested.

The 83 breeder colonies varied widely in their responses to tracheal mites. About two-thirds were statistically similar to the resistant reference and one-fourth were similar to the susceptible reference. Three queen producers had 30 of 31 breeder colonies that were classified as resistant. The other five queen producers had breeder colonies that were very variable and of which 40% were susceptible.

Levels of resistance to tracheal mites found in breeder colonies used by eight U.S. commercial queen producers.For each colony, the resistance index (RI) shows the colony resistance relative to that of a resistant reference stock (resistance index set at 0.0) and a susceptible reference stock (resistance index set at 1.0).The number of colonies of each operation that was tested appears immediately above the horizontal axis.

The most striking result of this survey was the variability in levels of tracheal mite resistance among colonies of U.S. commercial breeding stock. This breeding population can be expected to yield propagated queens that range widely in quality: some queens will be useful in improving stock by imparting resistance, while others will predispose their colonies to damaging mite infestations.In the absence of knowledge about the resistance levels of individual breeder colonies, the performance (vis-vis tracheal mites) of production colonies headed by commercial queens becomes largely a matter of the chance associated with a queen producer=s random selection of a grafting source from among his or her breeder colonies.

Fortunately, the majority of colonies we tested had useful resistance to tracheal mites. However, queens propagated from susceptible colonies and then widely distributed through commercial sales may contribute to the lingering problems associated with tracheal mites across the country.The findings from this survey emphasize the value of testing in enabling effective selection for resistance to tracheal mites.Through testing, susceptible colonies are easily identified and can be eliminated.Severalof the queen breeders who participated in this survey reported that susceptible colonies were removed from their breeding programs soon after they received the test results.

Although acaricides control varroa mites in colonies of honey bees, use of chemicals endangers bees and hive products.Bee breeders strive to reduce exposure of bees to chemicals by developing stocks of bees that resist the mites.Towards that goal we began breeding bees for resistance to varroa mites more than five years ago.Our project focused on finding varroa-resistance in honey bees from the U.S.

Initially, we found no bees that could survive varroa infestation without chemical control.Short field tests (Figure 1) were used to carefully measure growth of bee and mite populations in colonies that had genetically different queens.We defined resistance as the ability of a colony of bees to significantly limit growth of mite populations below the average colony.In any group of colonies, there is considerable variation in the rate of growth of mite populations.We hoped that small genetic differences between colonies of bees mediated differences in growth of mite populations.

We needed lines of bees that consistently and predictably limited the growth of varroa mite populations before identifying genetic traits related to resistance.Our strategy was to use queens from colonies of bees that significantly limited mite growth as breeder queens.Virgin queens and drones were raised from several different breeder queens.Then various combinations of drones and queens were made using instrumental insemination to control the matings (Figure 2).The newly inseminated queens were tested for varroa resistance in short field tests during the following season.The best queens were again chosen as breeders.The entire process was repeated through several generations until the ability to limit growth of varroa mite populations had been enhanced.

Because selection for varroa-resistance was based on overall mite growth, we knew little about the mechanism of resistance.All colonies started a field trial with the same mix of bees and mites. The only known differences were the test queens.We measured characteristics known to be associated with varroa-resistance (e.g. hygiene, grooming, reduced postcapping period, etc.) from all colonies during field trials.Then we searched for those traits that correlated best with the mite populations at the end of a test.

Step 1: A field test begins by collecting and mixing 50-70 lbs of bees from colonies that are not resistant to varroa mites. We choose colonies that have substantial populations of the varroa mites.

Step 2: The large mass of bees and mites is subdivided into smaller 500 gram units. Scoops of bees are added to pre-weighed cages. The cages are weighed again after bees are added to get an accurate estimate of the weight of the bees.

Step 3: Each test colony is given two cages of bees, a test queen, 4 combs and a feeder. The cages are paired so that each colony receives about 1 kg of mite-infested bees. The colonies remain closed (screen over entrances) for two days to minimize drift between colonies.

Step 4: Bee and varroa mite populations grow during an 80-115 day period when populations of bees and mites are estimated from each colony (see Measuring Mite Populations). We select colonies with the lowest mite growth as breeder queens.

Figure 1. Our typical set up of a field test. We try to ensure that all colonies are similar at start of the test. Typically, we use 25 colonies in a short test, and each colony begins the trial with ca. 1 kg of worker bees, 400 mites, and a queen bee. We measure mite and bee populations at the beginning and at the end of the test.

Although several characters predicted mite growth, the percentage of non-reproducing mites (%NR) correlated the most strongly with mite population growth. Female varroa mites reproduce within the capped brood cells of the honey bee (see Varroa Reproduction). Non-reproducing mites are those that enter brood cells to reproduce and either do not lay eggs, or if they do lay eggs, none of the daughters can mature before the adult bee leaves the brood cell. We define several types of non-reproducing mites: (a) mites that die before laying eggs, (b) live mites that do not lay eggs, (c) mites that produce only a son, (d) mites that produce progeny that die before reaching adulthood, and (e) mites that produce progeny so late in the development cycle of the bee that they do not have enough time to reach adulthood.

Figure 2. A queen being instrumentally inseminated. This technique allows the bee breeder to control the genetics of colonies of bees. It is very difficult to control the natural mating process of bees, which occurs high in the air.

We open capped brood cells containing tan-colored pupae (Figure 3) to measure %NR. Usually, a varroa mite lays all of her eggs by this stage of bee development (see Varroa Reproduction).We evaluate 30 singly-infested brood cells from a colony to determine %NR.We decide a mite's reproductive success by identifying the sex and maturation of her offspring.If the numbers of daughters and their development are considered normal, the mother mite is normally reproductive.However, if her oldest female daughters are under-developed and will not reach adulthood in the remaining time of the host pupa's development, or if there are no progeny, the mother mite is non-reproductive.

Figure 3. Brood cells containing tan-colored pupae are examined to measure the percentage of non-reproducing mites. Non-reproducing mites do not produce mature daughters before the host bee leaves the brood cell. We examine mite families from 30 singly-infested cells per colony. Mite families are evaluated for the numbers and maturity of the female offspring to decide if the mother mite will succeed in producing at least 1 mature daughter during the time remaining in the metamorphic development of the bee pupa.

Nearly all colonies of bees have some non-reproducing mites.In several tests, mite growth was lowest in colonies with the highest %NR.We changed our selection strategy to concentrate on the %NR rather than on overall growth of the mite population.We knew that a genetic character in bees was somehow causing mites to become non-reproductive in some colonies.We call the trait 'suppression of mite reproduction' (SMR).Although we classify non-reproductive mites into 5 categories, only 2 of them are consistently associated with resistance to varroa in our resistant bees.These two categories are (1) living mites that do not lay eggs, and (2) dead mites that had laid no eggs.The dead mites are unusual because most of them are entrapped by the cocoon (a condition rarely seen in control, or susceptible colonies of bees).

We do not know exactly what causes mites to become entrapped by the cocoon or to simply not lay eggs upon entering a brood cell. These symptoms of abnormal mite reproduction become apparent only after 4-6 weeks of placing a queen with the SMR trait into a colony of bees. This delayed suppression of mite reproduction is called SMRD. A second type of mite suppression occurs within the first brood cycle of placing a queen into a colony of bees. The acronym for this immediate suppression of mite reproduction is SMRI. Although we have seen both types of suppression of mite reproduction, our work has focused on SMRD.

Experiments suggest that daughter mites raised in colonies with the SMRD trait are the mites affected by the bees (and not the original mites that start an experiment). The mites that do not lay eggs had low numbers of stored sperm in their spermathecae when compared to mites that reproduced normally (Figure 4). More than half of these mites had no sperm at all. Currently, we do not know if the low sperm counts result from lack of mating between mites (see Varroa Reproduction), or if the sperm transferred by males are non-viable and do not reach the spermatheca within the female.

a. An intact spermatheca removed from an adult female varroa mite (as seen under a compound microscope).

b. Gentle pressure will rupture the spermatheca and release the sperm for counting.

c. A single spermatozoon separated from the group of sperm.

Figure 4. Living mites that lay no eggs (blue bar) had lower numbers of stored sperm when compared to mites that produced normal families (green bar) (15-28 mites were examined per bar).

In the early years, the ?best? or most resistant colony had 35--40% non-reproducing mites, which was only slightly better than the 10-25% non-reproducing mites found in colonies of unselected or susceptible bees.Now, we routinely produce inbred resistant colonies that contain 60--100% non-reproducing mites (Figure 5).

Unselected or control bees

Bees with the SMR trait

Figure 5. Reproductive status of mites from susceptible and varroa-resistant colonies of bees after 50 days in a short field test. Resistant bees were produced from queens selected for the suppression of mite reproduction delayed (SMRD). Each pie graph represents 230-300 mites sampled from 10 different colonies. Key: entrapped (red) = mites that died by being entrapped by the cocoon; no eggs (blue) = living mites that laid no eggs; other (yellow) = other infertile mites; and normal (green) = reproductive mites that will likely produce at least 1 mature daughter.

We now have varroa-resistant stocks of bees inbred for the SMR trait, and these colonies greatly limit mite growth. The U.S. queen rearing industry is geared toward the production of naturally mated queens, which makes the production of commercial inbred resistant queens very unlikely (unless queens are mated in an isolated area such as an island). However, queen producers can readily produce hybrid queens. We found mite growth to be intermediate between resistant bees and susceptible bees when resistant queens are free-mated with susceptible drones (Figure 6). Although colonies with hybrid queens (resistant x control) had intermediate populations of mites, they had half the mites found in the susceptible controls. Hence, even hybrid queens should provide beekeepers a tangible level of resistance.

Figure 6. Final mite populations for 57 colonies having 3 types of queens after a 115 day field test (mean above each bar). Resistant queens were inbred for the SMRD trait (red). Resistant x Control were queens having the SMRD trait free-mated to drones that did not have the trait (yellow). Control queens were queens that lacked the SMRD trait mated to drones that also lacked the trait (blue). All colonies began the test with 0.9 kg of bees and about 650 mites.

Reproduction by varroa mites coincides with the pupal stages of honey bee metamorphosis, which occur within a capped brood cell. We focus on the relationship between varroa mites and worker bees. Female varroa mites live on adult bees when not reproducing. Adult male mites cannot live outside the capped brood cell. The following pictorial will show how the growth of mite populations depends on the availability of honey bee brood.

Metamorphosis of Honey Bees

Development of honey bees is similar to the metamorphosis in butterflies. The stages of development and the duration of each stage for a worker bee are given by the following sequence:

egg (3 days) --> larva & prepupa (8 days) --> pupa (9 days) --> adult

total development time = 19.5 - 20 days

The egg and early larval stages live in uncapped brood cells. The last two days of the larval stage, the pupal stage, and the first half-day of the adult stage occur beneath capped brood cells. The following description of honey-bee metamorphosis contains references of time in days. The reference point is the moment the egg was laid by the queen bee.

A queen bee and her court of attendant worker bees.

Step 1: Queen bee lays the egg.

Only one queen lives within a colony of honey bees. She lays up to 2,500 eggs per day. The worker bees care for the queen and the young brood.

A single egg is laid in a brood cell.

The life of a worker honey bee begins when the queen lays a fertilized egg onto the base of a worker-sized brood cell.

Worker egg in bottom of brood cell (ca. 120x magnification).

A young worker larva lying in a pool of brood food.

Step 2: A larva hatches from egg after three days.

Nurse bees feed brood food to the larva within minutes of hatching. Glands in the head of nurse bees secrete the liquid diet. The nurse bees continue to feed the larva until the cell is capped.

Step 3: Larva grows large on steady diet of brood food.

The worker larva sheds its skin (or molts) as it grows. The first four molts occur every 24 hours after the larva hatches. The fourth larval molt occurs by the end of day 7 when the larva occupies the entire floor of its brood cell. At 8 days the larva sends out chemicals that signal attendant worker bees to cap the brood cell. The final larval molt occurs on the 11-12th day (see below).

An old larva as seen on the 7-8th day of development, just before the cell is capped by adult bees.

Adult mite on abdomen of an adult worker bee (photo courtesy of Dr. Keith Delaplane; University of Georgia).

Varroa mites live on adult bees when not laying eggs.

Adult varroa mites live on adult bees when not reproducing. The average time spent on adult bees between reproductive cycles varies from a few days to more than a month. About 7 days is typical.

A mite begins her reproductive cycle by invading a worker cell about 0-18 hours prior to it being capped. A single mite may experience 3-8 reproductive cycles in her entire life, but the average number of cycles is 3.

Step 4: A mite invades the brood cell before it is capped.

A varroa mite invades a brood cell by running from the belly-side of a nurse bee into the cell opening. She runs down the cell wall and into the brood food beneath the bee larva. The mite becomes immobile while immersed in the brood food (she looks dead while in the jelly).

She breathes air with specialized tubes called peritremes. The peritremes are the thin, pale-colored tubes between the last two pairs of legs.

Immobile varroa mite in brood food. The third and fourth pairs of legs have been spread to show the two peritremes.

A brood comb showing capped worker cells.

Varroa mites remain immobile while immersed in brood food.

A varroa mite remains motionless until all of the brood food is eaten by the larva during her first 24-30 hours in the cell. Most mites wake up and begin feeding on the blood of the bee larva by the end of the 9th day.

Step 5: Bee larva spins a cocoon.

The bee larva will defecate soon after eating all of the brood food. Then she spins a cocoon using silk that is produced by glands within her mouthparts. The cocoon completely surrounds the larva when it is finished. The cocoon usually provides a barrier between the larva and the feces, but sometimes larvae will defecate after the cocoon is complete. Metamorphosis is completed within the protective cocoon.

The bottom half of the cocoon that lines a worker brood cell. The top half was torn during removal from the cell. The dark spots are dried feces.

A varroa mite that has been trapped between the cocoon and the cell wall (as seen from above after removing the bee larva). (back)

Sometimes mites get trapped!

If a varroa mite does not wake up before the bee larva spins the cocoon, the cocoon will be spun over the mite. Then the cocoon separates the mite from the larva. We call this condition of the mite 'entrapped by the cocoon'. Usually, very few varroa mites become entrapped by the cocoon; however, we have produced resistant bees that have high levels (> 25%) of entrapped mites (see Breeding Honey Bees to Suppress Mite Reproduction). An entrapped mite will die because it cannot feed.

Step 6: The bee larva becomes a prepupa.

The bee larva changes into a prepupa within hours of finishing the cocoon. The prepupa lies motionless in the brood cell as it prepares to shed the last larval skin. This stage lasts for nearly two days spanning the 10-11th days of bee development.

Most varroa mites begin to suck the blood of the host bee during the late larval to early prepupal stage. The mite pierces the body of the host with her mouthparts and sucks the blood like a tick might suck the blood from a dog. A varroa mite and her family usually feed from one wound.

The feeding site (or wound) is usually located in the lower 1/3 of the brood cell. Varroa mites tend to repeatedly defecate on the cell wall near the feeding site. The pile of mite feces is white.

A worker prepupa. Notice the pile of mite feces located on the cell wall (to the right of prepupa).

Metamorphic Development of Varroa Mites

The development of varroa mites is similar to the gradual metamorphosis exemplified by other mites and insects like grasshoppers. The different stages of development and the duration of each stage are given in the following sequences:

All stages of a varroa mite development occur within the capped brood cell. A typical female mite will lay one male egg and 4 female eggs during her reproductive cycle in worker brood. However, only the son and the 1 or 2 oldest daughters will reach adulthood before the honey bee emerges from the brood cell (see below).

The first egg from a varroa mite (next to the right side of the mite). It was removed from the mite under a microscope. The white ring around the mite is light reflecting from the glue that was used to anchor it for dissection.

Step 7: The body of the mite swells as the first egg develops.

The steady diet of blood from the prepupa provides the varroa mite the nutrition needed to make eggs. A mite's body swells as an egg matures within her ovary.

Step 8: The prepupa sheds the last larval skin.

The prepupa becomes a pupa at the end of the 11th day after it has shed the old larval skin. This molt is the 5th and final larval molt of the honey bee.

The skin sticks to the floor of the brood cell [ca. 80x magnification].

A varroa mite (center) with her first egg (upper left). The yellowish material below the mite is the shed larval skin from the prepupa, and the white material in the lower right of the cell is the mite feces.

Step 9: The mite lays her first egg.

The first egg is laid on the wall of the brood cell before the end of the bee's prepupal stage. Otherwise, the egg is laid in the first pupal stage (below). The first egg is almost always a male. The female mite will lay an egg every 30 hours over the next few days (usually not more than 5 eggs are laid in a worker brood cell).

Step 10: The prepupa becomes a pupa.

The head of the prepupa enlarges by the end of the 11th day, marking the beginning of the pupal stage of bee development. The tissues of the pupa will continue changing to form the adult insect. The most noticeable changes are an increase in pigmentation of the eyes and body as the pupa ages.

The first pupal stage of the worker bee begins on the 11-12th day. Neither the body nor the eyes of the pupa are pigmented.

The eyes of the bee pupa appear pale pink but its body remains white on the 13th day.

Step 11: The eyes of the pupa begin pigmentation.

The eyes become pigmented before other parts of the bee. There is no movement of legs, antennae or mouthparts during the early pupal stages.

Varroa mite family grows.

The adult mite continues to lay eggs. At this point, a typical family of varroa mites consists of the mother mite, a male protonymph, a female protonymph and another egg that will become a female mite.

The male protonymph as seen during day 12-13 of the honey bee's pupal development. This cell also contains two additional mite eggs (one above and one to the right of the male)

Pupa of worker bee on the 13-14th day of development.

Step 12: Eye pigments darken.

The compound eyes and the ocelli (the three small eyes at the center of the head) appear pink on the 14th day. The pupa still does not move its appendages.

In regards to the mite family, the male protonymph and the oldest female protonymph will molt to become deutonymphs during this stage of bee development. The second oldest daughter mite hatches from the egg to become a protonymph, and the 4th egg is laid by the mother mite.

Step 13: Eyes are purple on the 15th day.

The body is still white or slightly yellow, but some brown pigment appears in the antennae and mouthparts at this stage.

The purple-eyed bee pupa as seen on the 15th day of development.

A mite family as seen at the beginning of the 15th day of bee development. The four mite progeny are: (1) egg [below mother mite], (2) female deutonymph [lower left of mother mite], (3) male deutonymph [immediately above mother mite] and (4) a female protonymph [farthest above and to left of mother mite].

The core of the mite family is completed.

Although the mother mite can lay more eggs (and they sometimes do), none of those additional progeny will have time to mature into adult mites before the honey bee emerges from the brood cell. In fact, the egg (the 3rd daughter for this particular mother mite) in the picture to the left has only a 13% chance of reaching adulthood.

Step 14: The bee's body darkens.

The body of the bee has a yellowish tan appearance on the 16-17th day of development. The pigmentation of the antennae, mouthparts and legs increases. Some slight movements of the legs can be seen at this time.

A tan-colored pupa as seen on the 16-17th days of metamorphosis.

The body of the pupa as seen on the 16-17th day. Notice that the joints on the legs are pigmented. The white spots on the pupa's abdomen are mite feces.

By the end of the 17th day, the pupa has a dark tan or gray appearance.

Step 15: Body becomes more pigmented.

As the pupa's body darkens, movements of the legs and mouthparts are more frequent. The wing pads become gray-colored.

Step 16: The oldest mite progeny reach adulthood.

The son and first daughter will reach adulthood during the 17-18th days of the bee's metamorphosis. After molting from the deutonymph stage, the young adult mites are white. Their bodies begin to darken over the next few hours to days. Female mites will become as brown as their mothers, but adult males remain a light tan color.

The adult son and adult daughter mites will mate several times. The female mite stores 40-70 spermatozoa within her spermatheca, and she will use them later in life to fertilize eggs that she lays in a brood cell. Mating occurs near or on the pile of mite feces. Some scientists believe that the mite feces contains chemicals that attracts both sexes to the spot for feeding and mating. These mites do not have eyes and may depend on touch or smell to find each other.

A mite family as seen on the 17-18th day of the bee's development. The family consists of the mother mite, and her adult son and adult daughter (both located the farthest above the mother mite). The remaining two progeny are female deutonymphs.

Adult male and female mites that had just recently molted from the deutonymph stage. Their shed skins are the transparent or whitish objects located to the right of the two mites (the male skin is smaller and located near the top of the cell, and the female skin is touching the honey bee's old larval skin)

Varroa mites shed their skins twice during metamorphosis.

Varroa mites go through two molts as they mature. The first molt occurs when a protonymph becomes a deutonymph. The skin from this first molt is so small that it cannot be seen. The second molt occurs during the transition from deutonymph to adult. The skin from this molt can be readily seen in the brood cell.

Step 17: Bee pupa prepares for final molt.

The body color of the pupa becomes a dark gray or black on the 18-19th day. Movements of the legs are more prevalent during this period. The wings expand, and hair grows on the body.

Bee pupa on the 18-19th day of development.

Close-up of the two skins that can be found in a brood cell after the adult bee leaves the cell. The skin to the lower left is the old larval skin that was shed in step 8 (above). The skin in the upper right is the pupal skin that was shed as the pupa became an adult bee.

Step 18: Pupa molts and becomes an adult bee.

The pupa-to-adult molt of the honey bee occurs by the end of the 19th day. Several hours after the wings expand, the adult bee sheds the pupal skin. Movements of the legs can be vigorous at this time, which can injure any soft-bodied mites that exist in the brood cell (the adult male or immature female mites).

Step 19: The adult bee chews away cell cap.

The pupa-to-adult molt occurs about 12-20 hours before the adult bee emerges from the cell. The young bee expands her wings and finishes hardening her exoskeleton during this period of time. Her body movements are frequent and strong; therefore, immature mites are not likely to survive. Even adult male mites are vulnerable.

An adult worker bee chewing away the cell capping so that she can emerge from her brood cell.

An adult worker bee emerging from her brood cell on the 19-20th day. This bee has an adult mite riding on her thorax (it's hard to see in the photo, but the reddish disc on the thorax is a varroa mite).

Step 20: Adult bee exits the cell.

The young adult bee leaves her brood cell about 20 days after the egg is laid. The adult female mites ride the bee as she exits. In most cases, the mother mite and only 1-2 mature daughters will leave the brood cell. An average of 1.4 - 1.5 daughters per mother mites is typical for a population of mites.

The male mite usually remains in the brood cell. He will be killed and removed by nest cleaning bees that prepare newly vacated brood cells to receive another egg from the queen bee.

The feeding activities of varroa mites damage their host bees.

Often worker bees that have been parasitized by varroa mites are underweight and have deformed wings. Injuries from the feeding mites damage and weaken bees to the point that the wings do not expand properly. Varroa mites can also infect bees with dangerous viruses and bacteria.

An adult worker bee with damaged wings as the result of developing within a brood cell infected by varroa mites (photo courtesy of Dr. Keith Delaplane; University of Georgia).

Although varroa mites infect worker brood cells of our western honey bee, they prefer drone brood cells when available (early spring). Development of a drones is similar to that of a worker bees, but the overall development time for drones is 23-24 days. The capped period for drones is 14-15 days. The extra 2.5 days (relative to the capped period for worker bees) provides time for an additional 1-2 daughter mites to mature. A single mother mite can produce 3-5 mature daughters within a capped drone brood cell, but the average number of daughters per mite in drone cells is about 2-2.5. We do not allow drones to be raised in our experimental colonies when we are breeding for resistance to varroa. Our research has focused on breeding for resistance to varroa mites (see Breeding Honey Bees for Suppression of Mite Reproduction) by observing mite reproduction in worker brood cells only.

Population Measurements

Measuring Mite Populations

The total number of varroa mites in a colony of bees is determined by estimating the number of mites on adult bees and the number of adult mites within the capped brood cells. Our procedures for these measurements are as follows:

Mites on adult bees

Weigh the entire hive, equipment and bees. We screen the colony's entrance during the night before weighing so that bees cannot leave during weighing.

Weigh the hive equipment without the bees. We brush all of the bees from the hive body and combs into an empty box before re-weighing the empty hive equipment. Do not shake the combs if they are to be examined for mite reproduction because immature progeny mites can be killed in a brood cell when the pupa is rattled against them.

The difference in these two weights is the weight of the adult bees in the colony.

Scoop ca. 1,000 bees from the box (after mixing) and put them into a pre-weighed jar. The jar is re-weighed, and the difference gives the weight of bees within the sample.

Wash each sample of bees with 75% ethanol (Figure 1). The bees are washed over a sieve that allows varroa mites to filter through the mesh while retaining the adult bees. We rinse each sample until we get two consecutive washes that contain no mites. This procedure gives us an estimate of the number of mites per gram of bees. For example, if 30 mites were counted from 150 grams of bees, the estimate is 0.2 mites per gram of bees.

Figure 1.A sample of adult bees is washed from each colony to determine the mite load on the adult bee population. The sample of bees is shaken in a jar of alcohol and poured over a wire mesh. The wire screen retains the bees while dislodged mites flow to the pan below.

The total number of mites found on all adults bees is found by multiplying the total weight of bees (3) by the mites per gram estimate (5). For example, a colony with 3,000 grams of bees containing 0.2 mites per gram would have a total of 600 mites on all adult bees.

Mites in capped brood cells

Estimate the total area of capped worker brood cells in the colony. We use a 1 x 1 inch wire grid placed over the brood comb to estimate the total square inches of brood for each side of the comb (Figure 2). We only measure worker brood because we do not allow drone brood within our test colonies.

Figure 2. The area of capped brood being estimated with a wire grid.

Convert square inches of capped brood into number of cells of capped brood. There are 23.6 worker-sized brood cells per square inch of capped brood. Multiply the total brood area by 23.6 to convert the area to number of brood cells. For example, 185 square inches of worker brood equals 4,366 cells.

Estimate infestation rate of capped brood. We select two brood combs from each colony to estimate the number of mites per 100 capped cells. We choose one comb containing young capped brood (prepupae and white-eyed pupae) and the second comb containing older brood (purple-eyed and tan pupae). We open 50 brood cells from each side of a brood comb and count the number of varroa mites within each cell (Figure 3). Brood cells are opened along a straight horizontal line that bisects the brood patch along its middle. Only foundress (or adult females that entered the cell) mites (and not progeny) are counted in this estimate. If 56 foundress mites are found in a total of 200 cells, we report 28 mites per 100 brood cells or 0.28 mites per brood cell.

Figure 3. The varroa mite infestation rate for a colony of bees is found by examining 200 brood cells using a dissecting microscope (and a bright light source). Typically, we uncap 50 brood cells from each side of two combs. Cells are uncapped in a straight line across the center of the patch of capped brood.

Total mites in the capped brood is found by multiplying the total number of brood cells (2) by the infestation rate (3). For our current example, the total mites in the capped brood cells is (4,366 cells) x (0.28 mites per cell), or 1222 mites.

Total mites = (mites on bees) + (mites in brood)

= 600 + 1222 = 1822 mites

The SMR/VSH trait explained by hygienic behavior of adult bees

The SMR/VSH trait explained by hygienic behavior of adult bees

We produced varroa resistant honey bees by selectively breeding from colonies with high percentages of infertile mites (Breeding Honey Bees that Suppress Mite Reproduction). The heritable trait in bees which causes high percentages of infertile mites was termed “suppression of mite reproduction” (or SMR trait) because we thought that the bees were directly interfering with mite reproduction (Harris & Harbo, 2000, Apidologie 31: 689-699).A mechanism explaining the SMR trait has not been described, but Ibrahim and Spivak (2003, ABJ 144: 406) found that bees with the SMR trait were very hygienic and were able to remove varroa-infested pupae from capped brood cells.They also suggested that SMR bees may selectively remove pupae having reproductive mites.

Seventeen combs were obtained from 7 source colonies.Sources had an average infestation of 12 mites per 100 capped worker cells, and 71 % of the mites were reproductive.Each source provided at least 2 combs, one into each type of recipient colony.The infestation rate and percentage of infertile mites were measured for all transferred combs after 7-9 days in recipient colonies.

Analyses were restricted to cells infested by a single varroa foundress because few multiply infested cells were found (17 in a total of 329 infested cells).Fewer mites were found in combs that had been transferred to SMR colonies (Table).This suggests the hygienic removal of infested pupae by the adult bees with the SMR trait.SMR bees removed 91 % of pupae having reproductive mites and 58 % of pupae having infertile mites that had produced progeny.Pupae having infertile mites that had no offspring were apparently not removed (Table).Thus, the cue for varroa-sensitive hygiene by bees with the SMR trait is probably related to the presence of mite offspring (Figure).

Table – Comparison of mite populations between combs that were transferred into control and SMR colonies.Newly capped cells of naturally infested brood were transferred, and mite populations were evaluated 7-9 days later by examining cells containing pupae that were 3 days from emergence.

Variablea

Combs into control colonies

(n=8)

Combs into

SMR colonies

(n=9)

Comparison of Least Squares Means

t

dfb

Pr > |t|

Percentage of infertile mites

31 ± 3 %

78 ± 4 %

8.44

15

< 0.0001

No. reproductive mitesc

5.6 ± 0.6

0.5 ± 0.4

7.39

8.28

< 0.0001

No. infertile mites with progenyc, d

1.2 ± 0.2

0.5 ± 0.1

3.63

15

< 0.003

No. infertile mites without progenyc

1.0 ± 0.2

1.0 ± 0.2

0.20

12.4

> 0.8

No. dead mitesc, e

0.4 ± 0.1

0.2 ± 0.1

1.47

15

> 0.15

Total infested cellsc

8.1 ± 0.8

2.1 ± 0.6

5.94

15

< 0.0001

Footnotes for Table

aLeast squares mean ± SE for each group of combs were determined post ANOVA.The model included (1) type of recipient colony (fixed effect), (2) source of infested comb (random effect), and (3) type of recipient ´ source of infested comb (random effect).The model for the percentage of infertile mites was weighted by the total number of singly infested cells from each comb, and the models for all other variables were weighted by the total number of brood cells that were examined from a comb.

bDegrees of freedom were estimated using the Kenward-Roger method (Proc Mixed, SAS Institute).

cValues reported as number of mites per 100 capped worker brood cells.We examined 315 ± 35 and 563 ± 180 (mean ± SD) capped worker cells in each comb that had been transferred into control and SMR colonies, respectively.

dInfertile mites with progeny included (1) foundress mites that had only a son, and (2) those whose oldest daughter could not mature before the host bee emerged from the brood cell (see Martin, 1994, Exp. Appl Acarol 18: 87-100)